Protein thermal denaturation and matrix glass transition in different protein-trehalose-water systems

Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose-water systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T(g)) and the functional properties of the encapsulated protein (thermal denaturation, T(den)). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehalose-water solvent. Conversely, at low hydration, lower T(g) values are measured in the presence of proteins: the lack of water promotes sugar-protein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T(den) increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T(g) and T(den) is also observed, in line with previous results; this finding suggests that collective water-trehalose interactions, responsible for the glass transition, also influence the protein denaturation.